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Matrix Biology 27 (2008) 242 – 253

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Hyaluronidase activity is modulated by complexing with various

polyelectrolytes including hyaluronan
Brigitte Deschrevel ⁎, Hélène Lenormand, Frédéric Tranchepain, Nicolas Levasseur,
Trias Astériou, Jean-Claude Vincent
Laboratoire “Polymères, Biopolymères, Membranes”, UMR 6522 CNRS-Université de Rouen, 76821 Mont-Saint-Aignan Cedex, France
Received 20 June 2007; received in revised form 31 October 2007; accepted 1 November 2007

Abstract

Hyaluronidase (HAase) plays an important role in the control of the size and concentration of hyaluronan (HA) chains, whose biological
properties strongly depend on their length. Our previous studies of HA hydrolysis catalyzed by testicular HAase demonstrated that, whilst the
substrate–dependence curve has a Michaelis–Menten shape with a 0.15 mol L− 1 ionic strength, at low ionic strength (5 mmol L− 1), a strong
decrease in the initial hydrolysis rate is observed at high substrate concentrations; the HA concentration for which the initial rate is maximum
increases when the HAase concentration is increased. After examination of various hypotheses, we suggested that this could be explained by the
ability of HA to form non-specific complexes with HAase, which thus becomes unable to catalyze HA hydrolysis. In order to verify this
hypothesis, we first showed from turbidimetric measurements that HAase, like albumin, is able to form electrostatic complexes with HA. Albumin
then was used as a non-catalytic protein able to compete with HAase for the formation of non-specific complexes with HA, allowing HAase to be
free and catalytically active. The kinetic results showed that the HA–HAase non-specific complex inhibits HAase catalytic activity towards HA.
Depending on the albumin concentration with respect to the HAase and HA concentrations, albumin can either remove this inhibition or induce
another type of inhibition. Finally, the extent of such non-specific interactions between polyelectrolytes and proteins in HAase inhibition or
activation, in particular under in vivo conditions, is discussed.
© 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Keywords: Hyaluronan; Hyaluronidase; Bovine serum albumin; Hyaluronan–protein complex; Hydrolysis kinetics; Enzymatic inhibition; Enzymatic activation

1. Introduction
Hyaluronan (HA) is a linear high-molar-mass polysaccharide
composed of repeating disaccharide units of β(1,4)-D-glucuro-
nic acid β(1,3)-N-acetyl-D-glucosamine. Since its discovery in
the vitreous humor by Meyer in 1936, it has been well-
established that HA is widely distributed in the extra-cellular
matrix (ECM) of vertebrate tissues and that it is involved in
many fundamental biological processes such as cellular
adhesion, mobility, proliferation and differentiation (Catterall,
Abbreviations: BSA, bovine serum albumin; CD44, cluster of differentiation
44; ECM, extra-cellular matrix; HA, hyaluronan; HAase, hyaluronidase; HAS,
hyaluronan synthase; HN, hyaluronectine; MMP, matrix metalloproteinase;
TIMP, tissue inhibitor of matrix metalloproteinase.
⁎ Corresponding author. Tel.: +33 2 35 14 00 74; fax: +33 2 35 14 67 04.
E-mail address: brigitte.deschrevel@univ-rouen.fr (B. Deschrevel).
0945-053X/$ - see front matter © 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved.
doi:10.1016/j.matbio.2007.11.002
1995; Delpech et al., 1997; Laurent, 1987; Rooney et al., 1995;
Kennedy et al., 2002). Recently, it has been demonstrated that
the functions of HA strongly depend on chain size, and that HA
fragments and native HA may have opposite roles (Stern et al.,
2006). For example, HA fragments containing 4 to 25
disaccharides (1.6 103 to 104 g mol− 1) have an angiogenic
action on endothelial cells (West and Kumar, 1989a b), contrary
to native HA which is anti-angiogenic (Deed et al., 1997). In
addition, increased HA levels have been observed in response to
severe stress and in tumor progression and invasion (Stern,
2004). All this supposes a tight regulation of the HA metabolic
pathways.
HA synthesis occurs on the cytoplasmic face of the plasma
membrane and is catalyzed by HA synthases (HAS) (Weigel et al.,
1997). The polysaccharide is transported outside of the cell as it is
being synthesized (Prehm, 1984). Hyaluronidases (HAase)
 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
catalyze HA cleavage. In the human body, all HAases are of the
endo-β-N-acetyl-hexosaminidase type (EC 3.2.1.35) (Csóka et al.,
2001; Stern and Jedrzejas, 2006), which means that they catalyze
β(1,4) bond hydrolysis. In tumor tissues, both HA and HAase
concentrations are high (Bertrand et al., 1997; Lokeshwar et al.,
1996; Victor et al., 1997; Toole, 2002), HA being of rather low
molar mass (Kumar et al., 1989; Lokeshwar et al., 1997) which may
favor angiogenesis and thus cancer progression. In addition, HA
oligosaccharides induce proteolytic cleavage of the cancer cell
CD44 receptors and thus promote tumor cell migration (Sugahara
et al., 2003, 2006). Although the precise role of HASs and HAases
in the production and/or in the control of the size and concentration
of HA chains has not yet been elucidated, all of this suggests that
HAase may control the balance between long and short HA chains.
According to Mio and Stern (2002), regulation of the size and
concentration of HA chains is kinetically and energetically more
efficient through the catabolic rather than the anabolic pathway. In
fact, HAase could be present in tissues together with inhibitors
which may allow HAase to be rapidly activated or inactivated.
However, even though some HAase inhibition activities have been
detected in vivo, very little is known about their molecular structure
and mechanism of action in vivo (Mio and Stern, 2002).
We examined the kinetics of native HA hydrolysis catalyzed
by HAase (Vincent et al., 2003; Astériou et al., 2006) using
bovine testicular HAase as a model, since, like human HAase, it
catalyzes the hydrolysis of the β(1,4) bonds in HA. Using a
physiological-type ionic strength, i.e. 0.15 mol L− 1, bovine
testicular HAase obeys the Michaelis–Menten law: the initial
reaction rate is a hyperbolic function of the substrate concentra-
tion (Vincent et al., 2003). At low ionic strength (close to 5 mmol
L− 1), the behavior of the reactive system is totally different: for
increasing HA concentrations, the initial hydrolysis rate succes-
sively increases, reaches a maximum and then decreases to a very
low level, close to zero, at high substrate concentrations, instead
of reaching a plateau, as it does for a Michaelis–Menten type
enzyme (Astériou et al., 2002; Astériou et al., 2006). In addition,
the substrate-dependencies obtained for various HAase concen-
trations indicates that the atypical behavior of the HA/HAase
system observed at high substrate concentrations and at low ionic
strength also depends on the HAase concentration (Astériou et al.,
2006). While Bollet et al. (1963) have already observed such a
low hydrolysis rate at high HA concentrations with rat kidney
HAase in 0.10 mol L− 1 acetate buffer at pH 3.8, they did not
provide any explanation for these results. Similarly, Aronson and
Davidson (1967) mention that rat liver HAase in 0.10 mol L− 1
acetate buffer at pH 3.9 exhibits inhibition when the HA
concentration is higher than 1.6 g L− 1 and that this inhibition is
prevented by 0.15 mol L− 1 NaCl.
Our detailed study of the HA/HAase reactive system at low
ionic strength has led us to examine various hypotheses to explain
the atypical kinetic behavior (Astériou et al., 2006), such as an
inhibition by excess of substrate as well defined in basic enzy-
mology (Laidler, 1958). Among these hypotheses, the only one
which could explain all the experimental results is based on the
formation of non-specific complexes between HA and HAase
(Astériou et al., 2006). According to this hypothesis, in addition to
specific catalytic complexes, HA and HAase are able to form non-
243
specific complexes by electrostatic interactions. When HAase is
non-specifically complexed with HA, its catalytic activity is
suppressed. Thus, with a high ratio for the HA concentration over
the HAase concentration, the formation of non-specific com-
plexes leads to a decrease in the concentration of HAase able to
form catalytic complexes and so, to a decrease in the initial
hydrolysis rate. Conversely, with a low ratio for the HA concen-
tration over the HAase concentration, the concentration of HAase
able to catalyze the hydrolysis is high and the HA/HAase system
behaves as a Michealis–Menten system.
The ability of polysaccharides and proteins to form complexes
has been known for a long time and, in the case of HA, it was
shown more than 60 years ago. The Mucin Clot Prevention
(Robertson et al., 1940) and the turbidimetric methods (Kass and
Seastone, 1944; Dorfman and Ott, 1948) were developed to assay
HAase. In the latter method, HAase is assayed by measuring the
disappearance of the turbidity resulting from complexes formed
between long chains of HA and albumin under acidic conditions.
Since then, some investigations (Gold, 1980; Grymonpré et al.,
2001) have been devoted to the characterization of the complexes
formed between HA and the bovine serum albumin (BSA). For
example, Xu et al. (2000) studied the influence of pH on the
solubility of HA–BSA electrostatic complexes. Moreover, others
working on HAase report that serum proteins are able to enhance
the HAase catalytic activity. Gacesa et al. (1981) have shown that
among the serum proteins, albumin has the greatest effect.
According to Maingonnat et al. (1999), other proteins such as
hyaluronectin (HN), which is a hyaladherin (i.e. a protein able to
specifically interact with HA), hemoglobin and immunoglobulins
are also able to enhance HAase activity and so, to increase the
sensitivity of the HAase detection. However, HAase activity
enhancement depends on the protein concentration.
The atypical behavior of the HA/HAase system at low ionic
strength together with both the ability of albumin to form
complexes with HA and the effect of added proteins on the HAase
activity have led us to suggest that the addition of a non-catalytic
protein, such as BSA, induces a competition between HAase and
the non-catalytic protein for the formation of non-specific
complexes with HA. Consequently, this addition of the non-
catalytic protein could allow the concentration of free, and thus
catalytically active, HAase, to increase, leading to an increase in
the HA hydrolysis rate. In order to test this hypothesis, we chose
to use BSA as the non-catalytic protein. The results of this study
show how non-specific complexes between HA and HAase
inhibited the HAase catalytic activity towards HA and how,
depending on the BSA concentration with respect to the HAase
and HA concentrations, BSA either removed this inhibition or
induced inhibition of the hydrolysis of HA catalyzed by HAase.
2. Results
2.1. Existence of HA–protein electrostatic complexes
The formation of non-specific complexes between HA and the
non-catalytic protein has been studied by using spectrophoto-
metric measurements. Spectra of a 0.73 g L− 1 HA solution, a 1 g
L− 1 BSA solution and a 0.73 g L− 1 HA plus 1 g L− 1 BSA mixture
244 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
at low ionic strength and at physiological-type ionic strength, all
at pH 4, were carried out. For the sake of clarity, throughout the
paper, low ionic strength signifies that no salt was added in the
medium and that the ionic strength, mainly due to the buffer, was
close to 5 mmol L− 1 and, physiological-type ionic strength
signifies that 0.15 mol L− 1 salt were added to the medium and that
its ionic strength was thus close to 0.155 mol L−1, a value
corresponding approximately to what is usually called physiolo-
gical ionic strength. As expected (Xu et al., 2000), Fig. 1 shows
that at low ionic strength, the HA and BSA spectra were clearly
not additive; the spectrum of the mixture, with values of absor-
bance much higher than those corresponding to the sum of the HA
and BSA spectra, was characteristic of turbidity. Conversely, at
physiological-type ionic strength, the spectrum of the mixture
closely resembled to the sum of the HA and BSA spectra.
Absorbance at 400 nm of mixtures containing 0.73 g L− 1 HA
and 2 g L− 1 BSA were recorded over time. At pH 4 (Fig. 2), at
physiological-type ionic strength, the absorbance did not vary as a
function of time and was equal to the sum of the individual
absorbances of HA and BSA, while at low ionic strength, the
absorbance strongly increased in less than 1 min and then quickly
decreased and stabilized at a value close to 0.8. At physiological-
type ionic strength, we observed the same behavior at pH 5 (Fig. 2)
as at pH 4. At low ionic strength, the absorbance increase was
extremely low at pH 5 (Fig. 2) compared to that measured at pH 4.
The same experiments were performed with HAase instead of
BSA. At physiological-type ionic strength, at pH 4 as well as at pH
5, the absorbance increased a bit immediately after the addition of
HAase. and then it stayed nearly constant (Fig. 3). This increase
corresponded to the absorbance of the HAase solution. At low ionic
strength, the same behavior was observed at pH 4 and 5 (Fig. 3): the
absorbance increased, then decreased and finally tended to stabilize
at a value higher than that measured at physiological-type ionic
strength. However, the rates of absorbance increase and decrease
were lower at pH 5 than at pH 4.
Fig. 1. Spectra between 250 and 650 nm of a 0.73 g L− 1 HA solution, a 1 g L− 1
BSA solution and a 0.73 g L− 1 HA plus 1 g L− 1 BSA mixture at low ionic
strength and at pH 4.
In these experiments, turbidity indicated that non-specific
complexes had formed following mixing of HA and protein.
The fact that turbidity appeared only under low ionic strength
conditions indicated the electrostatic nature of the interactions
established between HA and protein. At physiological-type
ionic strength, the concentration of small ions was sufficiently
high to screen at least part of the electric charges borne by HA
and protein and thus prevented the formation of a suspension of
HA–protein complexes. With a pKa value close to 2.9 (Berriaud
et al., 1998; Cleland et al., 1982), at pH 4 and above, HA is a
polyanionic molecule. According to the isoelectric pH (pI)
value for BSA, estimated to be close to 5.0 (Peters, 1975; Wang
et al., 1996; Xu et al., 2000), the net charge of BSA is positive at
pH 4 and close to zero at pH 5, while the net charge of HAase,
whose pI is around 6.8 (Astériou, 2002), is positive at pH 4 as
well as at pH 5. Knowing the charge state of HA, HAase and
BSA according to the pH allows us to explain why, at low ionic
strength, HAase was able to form with HA complexes in
suspension at pH 5 as well as at pH 4, whereas BSA was able to
form such complexes only at pH 4. Therefore, in order to study
the effect of complex formation between BSA and HA on the
kinetics of HA hydrolysis catalyzed by HAase, kinetic expe-
riments were performed at pH 4 instead of pH 5, as was done in
our previous study (Astériou et al., 2006).
The major difference between the HA/BSA and the HA/
HAase systems is that HAase, contrary to BSA, is able to form
with HA specific complexes associated with its catalytic acti-
vity. In other words, the development of the composition of the
reaction medium as a function of time for the HA/HAase system
includes the formation of specific and non-specific complexes,
while for the HA/BSA system, this development is due only to
the formation of non-specific complexes. In the case of the HA/
HAase mixtures (Fig. 3), the absorbance increase was due to the
formation of non-specific complexes between HA and HAase.
The subsequent absorbance decrease was due to both the stabi-
lization of the heterogeneous system, as observed for the HA/
BSA system, and a progressive decrease in HA chain size
resulting from the catalytic activity of HAase. Indeed, it has
been reported (Meyer and Rapport, 1952) that short HA chains
(lower than 6 103–8 103 g mol−1) are not able to form with
proteins complexes in suspension. Thus, for the HA/HAase
mixtures at low ionic strength, the fact that the rate of absor-
bance decrease was higher at pH 4 than at pH 5 (Fig. 3) is in
complete agreement with the kinetic results according to which
the HAase-catalyzed HA hydrolysis rate is higher at pH 4 than
at pH 5 (results not shown).
2.2. Effect of BSA addition on the kinetics of the HA hydrolysis
catalyzed by HAase
In order to test the effect of BSA on the kinetics of HA
hydrolysis by HAase, a preliminary experiment was performed
at low ionic strength and at pH 4.85. The HA concentration was
0.73 g L− 1 and HAase was 0.5 g L− 1. The ratio of the HA and
HAase concentrations was high enough for non-specific com-
plex formation to occur, and low enough for enzymatic hy-
drolysis to be measurable. Fig. 4 shows that, as expected,
 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253 245

Fig. 2. Absorbance at 400 nm, as a function of time, of mixtures containing 0.73 g L− 1 HA and 2 g L− 1 BSA at low ionic strength (●) and at physiological-type ionic
strength (■), at pH 4 and pH 5.

enzymatic hydrolysis occurred but the reaction rate was very
low. After 50 min, a small volume of concentrated BSA solution
was added, so that the BSA was 2 g L− 1. We observed (Fig. 4)
that the addition of BSA gave rise to a strong and immediate
enhancement of the hydrolysis rate, elevated from 0.7 to
17 μmol L− 1 min− 1, or 25-fold. This result was in agreement
with our hypothesis based on the formation of non-specific
complexes between HA and proteins and on the existence of a
competition between BSA and HAase to form those complexes.
A more detailed study of the effect of BSA on the kinetics of the
HA hydrolysis catalyzed by HAase was thus carried out.
2.3. Influence of the BSA concentration on the kinetics of the
HA hydrolysis catalyzed by HAase
A series of HA enzymatic hydrolysis experiments were
performed with various BSA concentrations, at low ionic
strength and at pH 4. The concentration of HA in the reaction
medium was 0.73 g L− 1, that of HAase was 0.5 g L− 1 and the
BSA concentration ranged from 0 to 4 g L− 1. For each
condition, the experimental points, obtained from our improved
version of the Reissig assay (Astériou et al., 2001; Reissig et al.,
1955), were fitted by the bi-exponential model developed by
Vincent et al. (2003). The initial hydrolysis rate was then
calculated.
Fig. 5 gives the initial hydrolysis rate plotted against BSA
concentrations and demonstrates that three domains should be
considered with respect to the BSA concentration. In the first
domain, corresponding to BSA concentrations between 0 and
0.1 g L− 1, the initial hydrolysis rate remained very low. The
BSA concentration was thus too low for competition between
BSA and HAase to occur for non-specifically complexed HA;
the HA concentration was high enough to complex both HAase
and BSA. Under these conditions, the concentration of free and

Fig. 3. Absorbance at 400 nm, as a function of time, of mixtures containing 0.73 g L− 1 HA and 2 g L− 1 HAase at low ionic strength (●) and at physiological-type ionic
strength (■), at pH 4 and pH 5.
246 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
Fig. 4. HA reducing end concentration plotted against the reaction time for the HA
(0.73 g L− 1) hydrolysis catalyzed by HAase (0.5 g L− 1) in 5 mmol L− 1 phosphate
buffer at pH 4.85 and 37 °C. After 50 min of reaction, BSA was added to the
reaction medium; the BSA concentration in the reaction medium was 2 g L− 1.
catalytically active HAase, and thus the initial reaction rate, did
not increase. In the second domain, corresponding to BSA
concentrations between 0.1 and 0.5 g L− 1, the initial hydrolysis
rate greatly increased up to a maximum value. The initial
reaction rate was 25 times higher in the presence of 0.5 g L−1
BSA than without BSA. In that BSA concentration range, the
competition between BSA and HAase to form non-specific
complexes with HA occurred, led to an increase in the fraction
of free and catalytically active HAase molecules and thus, to an
increase in the initial hydrolysis rate: the higher the BSA con-
centration, the higher the concentration of free HAase and the
higher the initial hydrolysis rate. For a BSA concentration of
0.5 g L− 1, the concentration of free HAase reached its maxi-
mum value, which probably corresponded to a value close to the
total HAase concentration. In the third domain, corresponding
to BSA concentrations higher than 0.5 g L− 1, the initial
hydrolysis rate slowly decreased and, for a BSA concentration
equal to 4 g L− 1, reached a value slightly higher than that
obtained without added BSA. As the BSA concentration was
increased, HA complexed more and more with BSA. Thus,
although the concentration of free and catalytically active
HAase was maximum, the ability of HAase to form a specific
enzyme–substrate complex became less and less because of
steric hindrance, due to the increasing levels of complex forma-
tion between HA and BSA.
2.4. Influence of BSA on the substrate–dependence of the HA
hydrolysis catalyzed by HAase
HA hydrolysis experiments were performed with different HA
concentrations, ranging from 0 to 1.46 g L− 1, at low ionic
strength, at pH 4 and with a 0.5 g L− 1 HAase concentration. For
one series of experiments, the BSA concentration in the reaction
medium was 0.25 g L− 1, and for the other, no BSAwas added. For
each hydrolysis experiment, the initial reaction rate was deter-
mined from the kinetic curve obtained by fitting the experimental
points. The substrate–dependence curves (i.e. initial hydrolysis
rate plotted against HA concentration) obtained with 0.25 g L− 1
BSA and without added BSA are given in Fig. 6. In the absence of
BSA, the substrate–dependence curve obtained at pH 4 had the
same shape as that previously obtained at pH 5 (Astériou et al.,
2006): for increasing HA concentrations, the initial hydrolysis
rate increased, reached a maximum at an HA concentration of
0.37 g L− 1, then decreased and finally reached a value close to
zero for HA concentrations above 0.73 g L− 1. As mentioned
above, according to our hypothesis, the atypical behavior
observed at high substrate concentrations was due to non-specific
complex formation between HA and HAase, which suppresses
HAase enzymatic activity: the higher the HA concentration, the
lower the concentration of free and catalytically active HAase.
In the presence of 0.25 g L− 1 BSA, the initial hydrolysis rate
increased as the HA concentration increased, and reached a
maximum at an HA concentration close to 0.73 g L− 1. The
maximum initial reaction rate was thus obtained for a higher HA
concentration in the presence of 0.25 g L− 1 than in the absence
of BSA. For HA concentrations ranging from 0 to 0.73 g L− 1,
the higher the HA concentration, the higher the enhancement of
the initial hydrolysis rate resulting from addition of BSA. For
HA concentrations up to 0.37 g L− 1, even though there was free
and catalytically active HAase in the absence of BSA, the fact
that initial reaction rates were enhanced by addition of BSA
showed that, even in these cases, a portion of HAase was
complexed to HA and thus inactive. By forming complexes
with HA, the added BSA allowed HAase to be released and this
in turn increased the initial reaction rate. In fact, in the absence
of BSA, the increase in the initial rate was due only to the
increase in the substrate concentration; this increase in HA
concentration also led to a greater non-specific complex forma-
tion with HAase and thus to a decrease in the concentration of
active HAase. In the absence of BSA, for HA concentrations
Fig. 5. Initial hydrolysis rate plotted against the BSA concentration in the
reaction medium for the HA (0.73 g L− 1) hydrolysis catalyzed by HAase (0.5 g
L− 1) in 5 mmol L− 1 phosphate buffer, at pH 4 and 37 °C.
 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253 247

the presence of 1 g L− 1 of BSA, the initial reaction rate was

33 μmol L− 1 min− 1 and with 4 g L− 1, it was 17 μmol L− 1
min− 1. As expected, 1 g L− 1 of BSA clearly enhanced the initial
hydrolysis rate, compared to that obtained with 0.25 g L− 1 of
BSA or without BSA. This confirmed that the BSA concentra-
tion necessary to give rise to HAase release increased with
increasing HA concentrations. However, for a given HA con-
centration, to avoid HA becoming too greatly complexed with
BSA and thus unable to form catalytic complexes with HAase,
the BSA concentration must not be too high. This was partially
the case when using 4 g L− 1 of BSA: although the maximum
HAase was free, the high level of BSA complexed with HA
suppressed HAase catalytic activity.

2.5. Influence of BSA on the enzyme–dependence of the HA

hydrolysis catalyzed by HAase

Fig. 6. Influence of the HA concentration on the initial hydrolysis rate of the HA In order to study the influence of BSA on the enzyme–
hydrolysis catalyzed by HAase (0.5 g L− 1) in 5 mmol L− 1 phosphate buffer, at dependence curve of the HA hydrolysis catalyzed by HAase at
pH 4 and 37 °C. HA enzymatic hydrolysis performed in the absence (●) and in pH 4 and at low ionic strength, two series of experiments were
the presence of 0.25 g L− 1 (■) BSA.
performed with reaction media containing 0.73 g L− 1 HA. For
the first series, HAase concentrations ranged from 0 to 3 g L− 1
ranging from 0.37 to approximately 0.73 g L− 1, the increase in and no BSA was added, and for the second series, HAase
the substrate concentration was not sufficient to compensate for concentrations ranged from 0 to 2 g L− 1 and 1 g L− 1 of BSA was
the decrease in the active HAase concentration and thus, the added. At low ionic strength and without added BSA, as for the
initial reaction rate decreased. When 0.25 g L− 1 of BSA was substrate–dependence curve, an atypical shape was observed for
added, sufficient HAase was released to increase the initial the enzyme–dependence curve of HA hydrolysis catalyzed by
reaction rate. For HA concentrations ranging from approxi- HAase (Fig. 7). For HAase concentrations equal to 0.5 g L−1 and
mately 0.73 to 1.46 g L− 1, the addition of BSA still had a below, the initial hydrolysis rate remained very low and close to
positive effect on the initial hydrolysis rate, but this effect zero, instead of increasing with increasing HAase concentration
diminished as the HA concentration was increased and became as it should normally do. In good agreement with previous
nearly non-existent for an HA concentration of at least 1.46 g results, under these conditions, the ratio of the HA and HAase
L− 1. In that range of HA concentrations, the fact that, in the concentrations was too high for the concentration of free and
absence of BSA, the initial rate was close to zero, indicated that catalytically active HAase to be significant; HAase formed non-
nearly all HAase was non-specifically complexed to HA. Fig. 5 specific complexes with HA. Above an HAase concentration of
indicates that, with an HA concentration as high as 0.73 g L− 1,
0.5 g L− 1 HAase and 0.25 g L− 1 BSA, the portion of HAase
released was not maximum but was high. Thus, in the presence
of 0.25 g L− 1 of BSA, the maximum of the initial rate for the
substrate–dependence curve (Fig. 6) probably corresponded to
an HA concentration somewhat higher than 0.73 g L− 1. More-
over, with a total concentration of proteins (0.5 g L− 1 HAase
and 0.25 g L− 1 BSA) maintained at a constant level, increasing
the HA concentration above that corresponding to the maxi-
mum of the initial rate led to a progressive decrease in the
competition between BSA and HAase in forming non-specific
complexes with HA. Consequently, although all the BSA was
complexed to HA, the portion of released HAase decreased and
the concentration of free and active HAase tended towards zero
at an HA concentration at least equal to 1.46 g L− 1. In other
words, with an HA concentration at least equal to 1.46 g L− 1
and above, and an HAase concentration equal to 0.5 g L− 1, a
0.25 g L− 1 BSA concentration was not sufficiently high to
induce any release of HAase.
Fig. 7. Influence of the HAase concentration on the initial hydrolysis rate of the
In order to verify the last point, two additional experiments HA (0.73 g L− 1) hydrolysis catalyzed by HAase in 5 mmol L− 1 phosphate
were performed with 1 and 4 g L− 1 of BSA respectively, the HA buffer, at pH 4 and 37 °C. HA enzymatic hydrolysis performed in the absence
concentration being 1.46 g L− 1 and that of HAase 0.5 g L− 1. In (▲) and in the presence of 1 g L− 1 (▼) BSA.
248 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
0.5 g L− 1, the shape of the enzyme–dependence curve became
normal: the initial reaction rate linearly increased when the
HAase concentration was increased. With 0.73 g L− 1 HA, the
non-specific complex formation between HA and HAase had
reached its maximum level and thus, the HAase concentration
was sufficiently high for a portion of HAase to be free and active.
This portion of free HAase increased linearly with increasing the
HAase concentration, and so did the initial hydrolysis rate.
With an HA concentration of 0.73 g L− 1 and an HAase
concentration of 0.5 g L− 1, according to the results on Fig. 5, a
BSA concentration of 1 g L− 1 corresponded to a level of BSA
complexed with HA slightly higher than that necessary to obtain
the maximum concentration of free and active HAase. In other
words, with 0.73 g L− 1 HA, a concentration of BSA of 1 g L− 1
must be high enough for a maximum of HAase to be free and
catalytically active whatever the enzyme concentration used.
Fig. 7 shows that, as expected, in the presence of 1 g L− 1 BSA,
the enzyme–dependence curve had a normal shape: the initial
hydrolysis rate linearly increased as the enzyme concentration
was increased.
3. Discussion
The ability of proteins to form complexes with polyelectrolytes
such as polysaccharides has been recognized for a long time and
has various applications. The spectrophotometric results of the
present study indicated that, at low ionic strength and at pH 4,
BSA and HA formed non-specific complexes, as revealed by the
appearance of turbidity upon mixing these two species. In fact, the
ability of proteins to produce complexes with HA has been used
since 1940, when Robertson et al. (1940) described the Mucin
Clot Prevention method for HAase assay, which was based on a
precipitate formation when adding acidified serum to HA. In
1944, Kass and Seastone (1944) published an HAase assay
method in which addition of acidified horse serum to HA
solutions gave a turbid suspension. Later, Dorfman and Ott (1948)
suggested substituting horse serum albumin for serum. This
turbidimetric method was further developed (Tolksdorf et al.,
1949), introducing the definition of the turbidity reducing unit
based on the formation of a turbid solution when BSA is mixed
with HA at pH 3.8. It constitutes the current United States
Pharmacopeia XXII assay for HAase (US Pharmacopeia, 1990).
Moreover, our spectrophotometric results are in agreement with
those of Xu et al. (2000) who have shown that, under 10− 3 mol
L − 1 ionic strength, according to the pH and the BSA
concentration, HA and BSA may form soluble or insoluble
complexes. On the basis of these observations, the most important
result from our spectrophotometric measurements is that HAase
too was able to form non-specific complexes with HA. The fact
that both pH and ionic strength influence the formation of the non-
specific complexes between HA and BSA or HAase clearly
indicates the electrostatic nature of the interactions involved in
these complexes. In addition, the turbidimetric behavior of each
HA/protein system studied with respect to pH agrees with the pKa
value of HA and the pI values of the two proteins, the difference in
the behavior between the two proteins being related to the
difference between their pI values.
As mentioned above, at low ionic strength, at pH 4 as well as
at pH 5, HA and HAase form non-specific complexes. For a
given HAase concentration, the higher the HA concentration, the
lower the portion of free HAase, up to an HA concentration
above which the portion of free HAase became very close to
zero. Thus, when studying the substrate–dependence of the
HAase-catalyzed hydrolysis of HA under these conditions,
increasing the HA concentration consisted in both an increase in
the substrate concentration and a decrease in the portion of free
and catalytically active HAase. In fact, the substrate–depen-
dence curves obtained clearly showed that, when involved in a
complex with HA, HAase is unable to catalyze the enzymatic
hydrolysis. Indeed, if HAase in the non-specific complexes HA–
HAase was catalytically active, the initial hydrolysis rate should
not decrease and should not reach a value near zero at high HA
concentrations. The atypical shape of the substrate–dependence
curves can thus be explained as the result of two opposite effects
associated to the increase in the HA concentration: i) an increase
in the substrate concentration, which allows the initial reaction
rate to increase, and ii) a decrease in the concentration of the
catalytically active enzyme, which leads to a decrease in the
initial reaction rate. The fact that HAase in the non-specific
complex HA–HAase is catalytically inactive means that HA
may behave as an inhibitor of HAase in addition to being its
usual substrate. This original result brings up a question about
the value of the stability constant for the non-specific complex
HA–HAase as compared to that of the specific catalytic complex
HA–HAase. Indeed, the kinetic results suggest that the non-
specific complex is more stable than the specific one.
All the kinetic results of the present study show that BSA was
able to compete with HAase to form non-specific complexes
with HA, with, as a consequence, the release of free HAase
which thus regained its catalytic activity. This means that the
stability of the non-specific complex HA–BSA was higher than
that of the non-specific complex HA–HAase. In fact, as long as
the added BSA allowed the release of HAase by forming non-
specific complexes with HA, it induced an increase in the initial
rate of the HA hydrolysis catalyzed by HAase. Under these
conditions, BSA acted as an activator of HAase or, in other
words, it prevented the inhibition of HAase by HA. Conversely,
when the maximum HAase was released after the addition of
enough BSA, further addition of BSA led to an increase in the
level of BSA complexed with HA: the more BSA complexed
with HA, the lower the initial reaction rate, because the
complexed BSA hinders the formation of specific complexes
between HA and HAase. In these cases, BSA behaved as an
inhibitor of HAase. Thus, according to its concentration with
respect to the HA and HAase concentrations, BSA may act
either as an activator or as an inhibitor of HAase.
The effects of proteins on HAase activity have already been
reported. The first studies, reviewed by Mathews and Dorfman
(1955), date back to the 1940s and concern the influence of serum
on HAase activity. According to these studies, serum, whatever its
origin, inhibited HAase. As early as 1941, Hobby et al. (1941)
ascribed this inhibition to salt formation between serum albumin
and HA. Gacesa et al. (1981) noted that the extent of inhibition
was largely dependent upon the ratio of enzyme over serum
 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
quantities. These results may be explained by the fact that the total
protein concentration in serum was high compared to the HAase
concentration used, so that serum proteins, by allowing for a high
level of proteins complexed with HA, behave as HAase inhibitor.
Another explanation given for HAase inhibition by serum is the
presence in serum of an HAase inhibitor whose level is increased
in the sera of patients with cancer, liver disease and dermatolo-
gical disorders (Mio et al., 2000). Attempts to characterize this
serum inhibitor conclude that it is a high-molar mass thermolabile
glycoprotein requiring magnesium for its inhibiting activity (Mio
et al., 2000; Mathews and Dorfman, 1955; Kolarova, 1975) which
is observed at pH values above 5 (Kolarova, 1975; Gacesa et al.,
1981; Mio et al., 2000). The fact that this serum inhibitor has no
effect on Streptomyces HAase whilst it inhibits bovine testicular,
snake and bee venom HAases (Mio et al., 2000) indicates that its
inhibition activity is not by complexing to HA. This serum
inhibitor, identified by Mio et al. (2000) as belonging to the inter-
α-inhibitor family, may thus be a more specific HAase inhibitor
than other serum proteins and be an inhibitor whose activity may
directly concern HAase. The results reported by Fiszer-Szafarz
(1968) show that the sera of cancer patients have a greater
inhibitory activity towards HAase than normal sera. However, the
inhibition activity of cancerous sera is magnesium-independent.
In fact, the high serum protein contents used in this study suggest
that this inhibition could, at least partly, be due to a high level of
proteins complexed with HA.
At pH values below 5, Gacesa et al. (1981) observe a marked
enhancement of bovine testicular HAase activity upon addition
of serum to incubation mixtures. They further show that the
greatest activation effect is obtained by using BSA as well as
human serum albumin. Gold (1982) shows that both bovine
testicular and human liver HAases exhibit increased activity in
the presence of BSA at pH 4. More recently, Maingonnat et al.
(1999) report a dependence of HAase activity with respect to
protein concentration at pH 3.8 that is very similar to that shown
in the present study. Our experimental results allowed us to
confirm these authors regarding the origin of the inhibitory
effect of high protein content. However, this is not the case
concerning the activation effect of proteins. Indeed, according
to Maingonnat et al. (1999), the protein activation effect may be
due to the fact that HA can bind small amounts of proteins that
facilitate the opening of the HA random coil, thus facilitating
HAase accessibility to HA. But, if such is the case, the inhibi-
tion we observed at low ionic strength and at high substrate
concentration should not depend on enzyme concentration
(Asteriou et al., 2006). An interesting result of the study of
Maingonnat et al. (1999) is that the protein effect on the HAase
activity is observed not only with BSA, but also with human
serum albumin, immunoglobulins, hemoglobin, transferrin and,
interestingly, with HN which is a hyaladherin. It should be noted
here that the dissociation constant for the specific complex HA–
HN, equal to 10− 9 mol L− 1 (Maingonnat et al., 1999), is of the
same order of magnitude as those for the non-specific complexes
HA–BSA and HA–lysozyme, which are about 10− 9 and
10− 8 mol L− 1 respectively (Van Damme et al., 1994). Lysozyme
is present in cartilage (Van Damme et al., 1994) and albumin is a
major protein of synovial fluid (Scott et al., 2000), two areas
249
which are also rich in HA. Knowing that the interactions
involved in the complex HA–BSA (Xu et al., 2000) and in the
complex HA–lysozyme (Van Damme et al., 1994) are electro-
static, and that a charge patch able to accommodate an HA
decamer exists at the surface of human serum albumin (as
Grymonpré et al. (2001) have shown with integrated computer
modeling), we suggest that the binding of HA to albumin and
lysozyme might in fact be specific. Here, the specificity of the
binding would not be associated to 3D structural features or
specific sequences, but rather to the existence of such charge
patches on protein surfaces.
Non-specific (or maybe specific) interactions between poly-
electrolytes and proteins may thus be responsible for enzymatic
inhibition as well as activation behaviors. The inhibition
behaviors may result from the formation of two types of com-
plexes: i) polyanion–HAase complexes, which directly inacti-
vate HAase, and ii) HA–polycation complexes, which
indirectly inactivate HAase by hindering HAase accessibility
to HA. Many polyanions, such as glycosaminoglycans (heparin,
heparan sulfate, dermatan sulfate), HA derivatives (O-sulfo-
nated HA) and synthetic polyanions (poly(styrene-4-sulfonate))
are known to inhibit HAase (Mathews and Dorfman, 1955;
Aronson and Davidson, 1967; Girish and Kemparaju, 2005;
Toida et al., 1999; Isoyama et al., 2006). In the case of O-
sulfonated HA, part of its inhibition activity could be of the
competitive type (Toida et al., 1999). Indeed, such HA deri-
vatives may behave as substrates analogues in inhibiting
HAase. According to more recent studies, the inhibitory activity
of all these compounds does not involve their binding to the
catalytic site of the enzyme. In good agreement with our finding
about the non-specific complexes HA–HAase, Girish and
Kemparaju (2005) suggest that inhibition by these polyanions
might be due to the formation of non-specific electrostatic
complexes between HAase and the polyanions. This phenom-
enon is demonstrated in the case of heparin (Maksimenko et al.,
2001). Such enzyme inhibition by formation of non-specific
complexes between polyanion and enzyme has already been
documented, for phospholipase A2 (Melo and Ownby, 1999;
Diccianni et al., 1990), β-amylase and catalase, (Mathews and
Dorfman, 1955). Inhibition of HAase activity by formation of
HA–polycation complexes concerns, as mentioned above,
proteins such as albumin, but also, polycationic polysaccharides
such as chitosan. Denuziere et al. (2000) report that HA and
chitosan form non-specific electrostatic complexes which make
the substrate unavailable for HAase. Considering the activation
effect of low protein content, it might also be interesting to
investigate the possible activation effect of low chitosan
concentrations.
One of the more important questions, for which we can
already highlight some pertinent points for further research,
concerns the extent of inhibition and activation effects with
respect to HAase within the ECM. The ECM is rich in poly-
electrolytes such as proteins, glycosaminoglycans and other
polysaccharides. The two essential conditions are thus pH and
ionic strength. The ability of a protein and a polysaccharide to
form a non-specific complex is related to the pI and pKa values
of the two species respectively. Considering the diversity in the
250 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
ECM, one might expect that HAase as well as HA could be
involved in non-specific complexes at pH close to neutrality.
For example, Van Damme et al. (1994) show that HA and
lysozyme, a protein secreted into the ECM of cartilage, form
non-specific complexes at pH 7.5 and under 0.04 mol L− 1 ionic
strength. Such a low ionic strength is likely to exist in cartilage
because of the exclusion of small ions by its highly charged
proteoglycans (Van Damme et al., 1994). However, the
inhibition and activation effects associated with non-specific
complex formation probably exist under higher ionic strength.
Indeed, the activation and inhibition of HAase by proteins is
observed by Maingonnat et al. (1999) when using an ionic
strength close to 0.15 mol L− 1. Similarly, the activation of
HAase upon addition of BSA reported by Gacesa et al. (1981) is
measured with reaction mixtures containing 0.1 mol L− 1 NaCl.
Moreover, while the spectrophotometric measurements with
HA and BSA at pH 5 showed no turbidity, the strong activation
effect observed upon addition of BSA in the reaction mixture in
our preliminary experiment at pH 4.85 suggested that soluble
non-specific complexes may exist at pH close to 5 and have the
same effect as the complexes in suspension observed at pH 4.
The possible existence of HA–protein and/or polyelectro-
lyte–HAase complexes, including HA–HAase non-specific
complexes, in ECM tissue may play an important role in the
regulation of the HAase activity under normal and pathological
conditions. Indeed, such complexes would allow for HAase to
be present but inactive in ECM. This situation would corres-
pond to that suggested by Stern (2005), in which HAase must be
deposited in ECM together with its inhibitor in the same way as
matrix metalloproteinases (MMP) are deposited together with
tissue inhibitors of MMP (TIMP). If HAase inhibition is due to
the existence of non-specific complexes, HAase activity might
thus be restored by any processes able to dissociate those
complexes. Such processes might be a slight pH or ionic
strength variation associated with ion exchange or, in the case of
non-specific HA–HAase complexes, the presence of a protein,
which might be a hyaladherin, capable of forming an HA–
protein complex that would be more stable than the complex
formed with HAase.
The existence of HA–protein and/or polyelectrolyte–HAase
complexes in ECM tissue might also account for some of the
difficulties encountered in attempts to purify HAase. Indeed, it
is observed that for highly purified HAase preparations no
enzymatic activity is detectable in the absence of added proteins
(Maingonnat et al., 1999; Mathews and Dorfman, 1955).
According to our results, we may suggest that with these
purified HAase preparations, HAase undergoes non-specific
complex formation with HA and is thus inactivated. Of course,
it has been observed (Stern, 2005) that, in addition to being
present at exceedingly low concentration, when HAase is
purified, it requires the presence of detergents and protease
inhibitors to maintain its catalytic activity. These species are
needed to avoid enzyme denaturation. Thus, added BSA could
act by preventing HAase denaturation. However, our spectro-
photometric and kinetic results show that to be catalytically
active HAase should not undergo non-specific complex
formation with HA. Therefore, even though proteins such as
BSA could prevent enzyme denaturation by interacting with
HAase, their interaction with HA allows non-specific HA–
HAase complex formation to be avoided. Delpech (personal
communication) observes that preparations of HAase, which do
not show any enzymatic activity when they become highly
purified, have the same significant activity whether Triton X100
or albumin is added to the reaction mixture. On the basis of our
results, we may suggest that by interacting with HAase, Triton
X100 allows for HAase to be catalytically active by preventing
both HAase denaturation and HA–HAase complex formation.
Moreover, the ability of HA and HAase to form non-catalytic
complexes with each other and with various polyelectrolytes
might also explain why no enzymatic activity has yet been
detected in HYAL3 and HYAL4 gene products. Considering
these phenomena of complex formation might be useful in
designing an appropriate HAase activity assay method for
detecting new HAase activity.
Finally, although biochemists, in an attempt to identify a
single function for biomacromolecules such as enzymes are
making great efforts to purify them to homogeneity, the present
study shows that biomacromolecules may not have just one
biological function. In addition, interactions between the bioma-
cromolecules themselves may modulate their functions. This
often neglected fact should be kept in mind when attempting to
elucidate the biological functions of biomacromolecules and
also when therapeutic uses of these molecules are to be
considered.
4. Experimental procedures
4.1. Preparation and characterization of the HA solution
Sodium HA from human umbilical cord was obtained from
Sigma (H 1876, lot 127H0482). An HA stock solution was
prepared by dissolving HA in 5 mmol L− 1 phosphate (Prolabo
28 015.294) buffer, then it was divided into fractions and stored
P
at − 20 °C before use. The HA average molar mass ( M n ) and its
polydispersity index (Ip) were determined by steric exclusion
HPLC equipped with both multi angle laser light scatterring and
P
refractive index detectors (Tranchepain et al., 2006): (M n ) was
6 −1
equal to 1.45 10 g mol and Ip equal to 1.8. HA being highly
hygroscopic, we used the uronic acid assay method (Dische,
1947; Bitter and Muir, 1962) to measure the actual HA content
of the HA mother solution. The experimental procedure was as
described previously (Vincent et al., 2003). The method was
calibrated using sodium glucuronate (Sigma G 8645). The HA
stock solution contained 7.3 g of HA per litre.
4.2. Preparation of the HAase and BSA solutions
Bovine testicular HAase (EC 3.2.1.35) and BSA were from
Sigma and were used without further purification. HAase
(Sigma H 3884, lot 76H8025) had a specific activity of 990 units
(US Pharmacopeia, 1990) per mg. Since the enzymatic activity
of frozen HAase solutions did not remain constant, solutions
freshly prepared by dissolving HAase in 5 mmol L− 1 phosphate
buffer were used. Similarly, concentrated BSA solutions were
 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253
freshly prepared by dissolving BSA (Sigma A 3675, lot
78H1399) in 5 mmol L− 1 phosphate buffer.
4.3. Spectrophotometric measurements
The HA and protein solutions were diluted with 5 mmol L− 1
phosphate buffer. When necessary, before pH adjustment with
phosphoric acid (Sigma P 6560), an appropriate volume of a
concentrated sodium nitrate (Prolabo 27 950.298) solution was
added to the HA and protein solutions to adjust ionic strength. For
ionic strength adjustment of the solutions used to perform spectra,
potassium chloride was substituted for sodium nitrate because of
the absorbance of the latter between 250 and 350 nm. All the
solutions were preincubated at 37 °C. When the HA/protein
mixtures were analyzed, the HA solution was first placed in the
cuvette and the addition of the protein solution corresponded to
time zero for data recording. Quartz cuvettes of 1 cm pathlength
were used to obtain the spectra of the HA, BSA and HA plus BSA
solutions. For measurements of the absorbance at 400 nm over
time, the HA/protein mixtures were placed in a 1 cm pathlength
plastic cuvette. In all cases, the cuvette was placed in the spec-
trophotometer (Uvikon 860, Kontron) equipped with a magnetic
stirrer and maintained at 37 °C.
4.4. Kinetics of the HA hydrolysis
The HA stock solution was placed in a reactor, diluted to the
desired concentration with 5 mmol L− 1 phosphate buffer, ad-
justed to pH 4 with phosphoric acid, stirred and maintained at
37 °C. An appropriate volume of a concentrated BSA solution
adjusted to pH 4 was then added to the HA solution. For the
experiments performed in the absence of BSA, the BSA was
replaced by phosphate buffer at pH 4. The reaction was started
2 min after the addition of BSA, by adding an adequate volume
of a concentrated HAase solution previously adjusted to pH 4.
The BSA and HAase solutions were preincubated at 37 °C.
According to Gacesa et al. (1981) and Gold (1982), the fact
that the enzyme is preincubated with or without the non-
catalytic protein (serum, serum proteins or albumin) for several
hours before mixing with HA does not significantly modify
enzyme activity means that the non-catalytic protein does not
act by preventing enzyme inactivation. By taking into account
our hypothesis, we chose to preincubate BSA with HA, thus
allowing for BSA to complex with HA and under the expe-
rimental conditions used, for the maximum HAase to be free
and catalytically active. We have tested the influence of the
preincubation time of BSA with HA: no significant differences
in the initial hydrolysis rate were observed for preincubation
times ranging from 0 to 20 min and a preincubation time of
2 min was retained.
Throughout the reaction, 200 μL aliquots of the reaction
medium were removed from the reactor and assayed with the N-
acetyl-D-glucosamine reducing ends assay. The concentration of
N-acetyl-D-glucosamine reducing ends, generated from HA
hydrolysis, was measured by using our improved version
(Astériou et al., 2001) of the Reissig method (Reissig et al.,
1955) according to the experimental procedure detailed in
251
Vincent et al. (2003). This improved version allows the turbi-
dimetric component, due to the presence of proteins in the
reaction medium samples, to be deduced from the total absor-
bance measured at 585 nm, thus giving the colorimetric com-
ponent of the absorbance. This color part of the absorbance at
585 nm (Abs585) is related to the molar concentration of N-
acetyl-D-glucosamine reducing ends, which also corresponds to
the molar concentration of HA reducing ends ([HA reducing
ends]), according to the following equation:


½HA reducing ends ¼ Abs585 = 1:8103  1:63
where 1.8 · 103 corresponds to the slope of the calibration curve
obtained by using N-acetyl-D-glucosamine (Sigma A 8625) and
1.63 is a corrective factor due to the fact that in HA, N-acetyl-
glucosamine is substituted in position 3 (Shimada and
Matsumura, 1980; Vincent et al., 2003).
For each kinetic experiment, the hydrolysis reaction was
followed for 3 h and the concentration of the HA reducing ends
was plotted against time. The experimental points obtained were
fitted by the bi-exponential model described in Vincent et al.
(2003) and the initial hydrolysis rate was calculated as being
equal to the value of the first derivative of that function at time
zero.
Acknowledgements
We thank Dilys Moscato for reading the manuscript. We are
grateful to the “Conseil Régional de Haute-Normandie” for the
fellowship granted to Trias Astériou, to the “Matériaux Poly-
mères, Plasturgie” network for the fellowship granted to Frédéric
Tranchepain and to the French Research and Technology Ministry
for the fellowship granted to Hélène Lenormand.
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